Template-switching of CMV-RNA at replication.

以前我们已经证明在侵染烟草叶片的黄瓜花叶病毒(CMV)中存在卫星RNA的多聚体。在本研究中，我们首先假设在感染病毒的植物中合成了异源连接的RNA分子，例如CMV卫星RNA与CMV基因组RNA连接，根据CMV基因组和卫星RNA的核苷酸序列设计寡核苷酸引物来扩增头到尾的异源连接分子中的连接区。从侵染CMV的烟草叶片中提取总RNA，经聚合酶链式反应(PCR)扩增出全长的c DNA。对RT-PCR产物的序列分析表明，它们是CMV基因组RNA1-3之间的正义或负义连接RNA，而不是正负连接RNA。连接序列分析表明，大多数分子缺少3‘组分序列，少数分子在5’和3‘组分之间有插入。然而，从纯化的CMV制备物中没有检测到头尾相连的RNA。我们还从感染黄瓜绿斑驳花叶病毒(CGMMV)的样本中检测到正义或负义头尾相连的RNAs。这些数据表明，异源连接的重要RNA分子是由复制酶驱动的复制选择机制产生的。在本研究中，我们成功地建立了检测病毒RNA的简单直接管RT-PCR方法，并构建了具有感染性的CMV全长cDNA克隆，以确定病毒感染标本中的头尾相连RNA。

Previously we have showed that multimers of satellite RNA are present in cucumber mosaic virus (CMV) infecting tobacco leaves. First of all in this research we assumed that the hetero-joined RNA molecules were synthesized in virus-infected plants, for example CMV satellite RNA joined with CMV genomic RNA, oligonucleotide primers were designed on the basis of the nucleotide sequence of CMV genome- and satellite RNAs to amplify the junction region in head-to-tail hetero-joined molecules. The cDNA was synthesized from total RNA extracted from CMV-infecting tobacco leaves and amplified by PCR with the primers. Sequence analysis of RT-PCR products revealed that they were plus-sense or minus-sense head-to-tail joined RNAs between CMV genomic RNA1-3 but not plus-minus joined one. The junction sequences indicated that most molecules lacked sequence of 3' components and a few have insertion between 5' and 3' components. However, no head-to-tail joined RNA was detected from purified CMV preparation. We have also detected plus-sense or minus-sense head-to-tail joined RNAs from samples infected with cucumber green mottle mosaic virus (CGMMV). These data indicate that hetero-joined vital RNA molecules are generated by a replicase-driven copy-choice mechanism. In this research we have succeeded to develop the simple direct tube RT-PCR method that can detect virus RNA.Moreover, to confirm the head-to-tail joined RNAs from virus-infecting samples, the infectious full-length cDNA clone of CMV was constructed.